This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding serine O-acetyltransferase in plants and seeds.
Sulfate assimilation is the process by which environmental sulfur is fixed into organic sulfur for use in cellular metabolism. The two major end products of this process are the essential amino acids cysteine and methionine. These amino acids are limiting in food and feed; they cannot be synthesized by animals and thus must be acquired from plant sources. Increasing the level of these amino acids in feed products is thus of major economic value. Key to that process is increasing the level of organic sulfur available for cysteine and methionine biosynthesis.
Multiple enzymes are involved in sulfur assimilation. These include high affinity sulfate transporter and low affinity sulfate transporter proteins which serve to transport sulfur from the outside environment across the cell membrane into the cell (Smith et al. (1995) PNAS 92(20):9373-9377). Once sulfur is in the cell, sulfate adenylyltransferase (ATP sulfurylase) (Bolchia et al. (1999) Plant Mol. Biol. 39(3):527-537) catalyzes the first step in assimilation, converting the inorganic sulfur into an organic form, adenosine-5xe2x80x2 phosphosulfate (APS). Next, several enzymes further modify organic sulfur for use in the biosynthesis of cysteine and methionine. For example, adenylylsulfate kinase (APS kinase) catalyzes the conversion of APS to the biosynthetic intermediate PAPS (3xe2x80x2-phosphoadenosine-5xe2x80x2 phosphosulfate) (Arz et al. (1994) Biochim. Biophy. Acta 1218(3):447-452). APS reductase (5xe2x80x2 adenylyl phosphosulphate reductase) is utilized in an alternative pathway, resulting in an inorganic but cellularly bound (bound to a carrier) form of sulfur (sulfite) (Setya et al. (1996) PNAS 93(23):13383-13388). Sulfite reductase further reduces the sulfite, still attached to the carrier, to sulfide and serine O-acetyltransferase converts serine to O-acetylserine, which will serve as the backbone to which the sulfide will be transferred to from the carrier to form cysteine (Yonelcura-Sakakibara et al. (1998) J. Biol. Chem. 124(3):615-621 and Saito et al. (1995) J. Biol. Chem. 270(27):16321-16326).
As described, each of these enzymes is involved in sulfate assimilation and the pathway leading to cysteine biosynthesis, which in turn serves as an organic sulfur donor for multiple other pathways in the cell, including methionine biosynthesis. Together or singly these enzymes and the genes that encode them have utility in overcoming the sulfur limitations known to exist in crop plants. It may be possible to modulate the level of sulfur containing compounds in the cell, including the nutritionally critical amino acids cysteine and methionine. Specifically, their overexpression using tissue specific promoters will remove the enzyme in question as a possible limiting step, thus increasing the potential flux through the pathway to the essential amino acids. This will allow the engineering of plant tissues with increased levels of these amino acids, which now often must be added a supplements to animal feed.
The present invention concerns isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide having serine O-acetyltransferase activity wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:3 have at least 90% sequence identity, or wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:5 have at least 85% sequence identity. It is preferred that the sequence identity to SEQ ID NO:5 be at least 90%, it is more preferred that the sequence identity to SEQ ID NO:3 or to SEQ ID NO:5 be at least 95%. The present invention also relates to isolated polynucleotides comprising the complement of the nucleotide sequence. More specifically, the present invention concerns isolated polynucleotides encoding the polypeptide sequence of SEQ ID NO:3 or SEQ ID NO:5 or nucleotide sequences comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4.
In a first embodiment, the present invention relates to an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 200 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:3 have at least 90% or 95% sequence identity based on the ClustalV alignment method, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 250 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:5 have at least 85%, 90% or 95% sequence identity based on the ClustalV alignment method, or (c) the complement of the first or second nucleotide sequence, wherein the complement and the first or second nucleotide sequence contain the same number of nucleotides and are 100% complementary in a pairwise alignment. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:3, and the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:5. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, and the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:4. The polypeptide preferably has serine O-acetyltransferase activity.
In a second embodiment, the present invention concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence, and a cell, a plant, and a seed comprising the recombinant DNA construct.
In a third embodiment, the present invention relates to a vector comprising any of the isolated polynucleotides of the present invention.
In a fourth embodiment, the present invention concerns an isolated polynucleotide comprising a nucleotide sequence comprised by any of the polynucleotides of the first embodiment, wherein the nucleotide sequence contains at least 30, 40, or 60 nucleotides.
In a fifth embodiment, the present invention relates to a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention, and the cell transformed by this method. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.
In a sixth embodiment, the present invention concerns a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a plant from the transformed plant cell. The invention is also directed to the transgenic plant produced by this method, and seed obtained from this transgenic plant.
In a seventh embodiment, the present invention relates to an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 200 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:3 have at least 90% or 95% sequence identity based on the ClustalV alignment method, or (b) a second amino acid sequence comprising at least 250 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:5 have at least 85%, 90%, or 95% sequence identity based on the ClustalV alignment method. The first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:3, and the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:5. The polypeptide preferably has serine O-acetyltransferase activity.
In an eighth embodiment, the invention concerns a method for isolating a polypeptide encoded by the polynucleotide of the present invention comprising isolating the polypeptide from a cell containing a recombinant DNA construct comprising the polynucleotide operably linked to at least one regulatory sequence.
In a ninth embodiment, the present invention relates to a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the recombinant DNA constructs of the present invention.
In a tenth embodiment, the invention concerns a method of selecting an isolated polynucleotide that affects the level of expression of a gene encoding a serine O-acetyltransferase protein or activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; (b) introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; (c) measuring the level of serine O-acetyltransferase protein or activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of serine O-acetyltransferase protein or activity in the host cell containing the isolated polynucleotide with the level of serine O-acetyltransferase protein or activity in the host cell that does not contain the isolated polynucleotide.
In an eleventh embodiment, the invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a serine O-acetyltransferase protein, preferably a plant serine O-acetyltransferase protein comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a serine O-acetyltransferase protein amino acid sequence.
In a twelfth embodiment, this invention concerns a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a serine O-acetyltransferase protein comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In a thirteenth embodiment, this invention relates a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the recombinant DNA construct of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the serine O-acetyltransferase polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a fourteenth embodiment, this invention concerns a method of altering the level of expression of a serine O-acetyltransferase protein in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the serine O-acetyltransferase protein in the transformed host cell.